Background: Hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiovascular disorder characterized by a combination of genetic and phenotypic heterogeneity. This study aimed to identify potential biomarkers and therapeutic targets for HCM using bioinformatics and animal models. Methods: Differentially expressed genes (DEGs) were identified from the GSE180313 and GSE160997 datasets using R programming. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed key biological functions and pathways related to HCM. A protein-protein interaction (PPI) network was built using the STRING database and Cytoscape software to identify hub genes. Additionally, drug prediction and molecular docking simulations were conducted to identify potential therapeutic agents. An HCM model was established in mice using intraperitoneal doxorubicin (DOX) injections, RT-qPCR and western blot employed to validate hub genes. Results: A total of 101 overlapping DEGs were identified, including 44 up-regulated and 57 down-regulated genes. Seven hub genes, including SERPINE1, showed significant variance in expression across the GSE261129 and GSE206978 datasets. Candidate drugs, especially Cetrorelix, had the strongest binding to SERPINE1, while standard HCM drugs showed weaker interactions. Cardiac dysfunction and myocardial injury were increased in the HCM group compared to the CON group. In particular, SERPINE1, FN1, C3, GRIP1, and F2R mRNA levels were up-regulated, while ACE and SYN2 were down-regulated in the HCM group. Protein levels of hypertrophy markers (Anp, Bnp), SERPINE1, and apoptosis markers were also elevated. Conclusions: The study identified core genes that are potentially involved in the pathogenesis of HCM, with SERPINE1 emerging as a novel therapeutic target for HCM.